2-Aminopurine as a fluorescent probe for DNA base flipping by methyltransferases.
DNA base flipping, which was first observed for the C5-cytosine DNA methyltransferase M. Hha I, results in a complete removal of the stacking interactions between the target base and its neighbouring bases. We have investigated whether duplex oligodeoxynucleotides containing the fluorescent base analogue 2-aminopurine can be used to sense DNA base flipping. Using M. Hha I as a paradigm for a base flipping enzyme, we find that the fluorescence intensity of duplex oligodeoxynucleotides containing 2-aminopurine at the target site is dramatically enhanced (54-fold) in the presence of M. Hha I. Duplex oligodeoxynucleotides containing 2-aminopurine adjacent to the target cytosine show little fluorescence increase upon addition of M. Hha I. These results clearly demonstrate that duplex oligodeoxynucleotides containing 2-aminopurine at the target site can serve as fluorescence probes for base flipping. Another enzyme hypothesized to use a base flipping mechanism is the N6-adenine DNA methyltransferase M. Taq I. Addition of M. Taq I to duplex oligodeoxynucleotides bearing 2-aminopurine at the target position, also results in a strongly enhanced fluorescence (13-fold), whereas addition to duplex oligodeoxynucleotides containing 2-aminopurine at the 3'- or 5'-neighbouring position leads only to small fluorescence increases. These results give the first experimental evidence that the adenine-specific DNA methyltransferase M. Taq I also flips its target base.
Holz
B
B
Max-Planck-Institut für Molekulare Physiologie, Abteilung Physikalische Biochemie, Rheinlanddamm 201, D-44139 Dortmund, Germany.
Klimasauskas
S
S
Serva
S
S
Weinhold
E
E
D015075:2-Aminopurine / Q000737:chemistry; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D015075:2-Aminopurine; D004247:DNA; C037664:DNA modification methylase HhaI; C059680:DNA modification methylase TaqI; D015257:DNA-Cytosine Methylases; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)